Using differential display PCR, we have identified two novel transcripts that are induced as a prechondroblastic cell line, MLB 13 MYC clone 17, acquires markers of the osteoblast phenotype in response to BMP-2 treatment. The first gene encodes a novel kinase that, when stably expressed in MC3T3E1 cells, results in a dramatic attenuation of the program of osteoblast differentiation. The second gene encodes a new member of the WD40 repeat family of proteins that markedly accelerates the program of osteoblast differentiation when stably expressed in MC3T3E1 cells. The presence of 7 WD repeats and of a short amino terminal non-beta-propeller region, coupled with the absence of a carboxy terminal extension and of a putative transactivation domain, link this protein most closely to the beta-transducin subfamily. However, the lack of a coiled-coil structure in the N- terminal extension of BIG-3 (BMP-2 Induced Gene-3Kb), and its cytoplasmic location, support the hypothesis that this protein is not a beta-transducin homolog. The studies proposed will address the hypothesis that the effects of BIG-3 on osteoblast differentiation require other actions of BMPs, by examining the program of osteoblast differentiation in noggin-treated MC3T3-E1 cells stably transfected with BIG-3 or empty vector. To address whether BIG-3 is essential for osteoblast differentiation, BIG-3 protein synthesis, in untransfected MC3T3-E 1 cells, will be blocked using antisense RNA strategies. Inhibition of BIG-3 protein expression will be evaluated and the impact of this reduction in BIG-3 protein levels on the differentiation of MC3T3-E1 cells will be examined. Deletion analyses will be performed to address the hypothesis that the amino terminal, non-WD repeat sequences contribute both to subcellular location and to the function of BIG-3. Yeast two-hybrid analyses will be performed to isolate proteins that interact with BIG-3 and contribute to its effects on osteoblast differentiation. Transgenic mice overexpressing BIG-3 in early osteoblasts (Col1-BIG-3) will be generated to address the hypothesis that BIG-3 accelerates the program of osteoblast differentiation in vivo. Analyses will be performed to assess whether this is associated with a parallel increase in osteoclast activity or whether uncoupling of bone formation and bone resorption is observed in the Col1-BIG-3 mice. Studies in primary osteoblasts isolated from these mice will determine if the overexpression of BIG-3 accelerates the differentiation of primary osteoblasts and whether it has effects on the differentiation and number of "osteoprogenitors" that give rise to endochondral and intramembranous bone.